This U.S. non-provisional application claims priority under 35 U.S.C. §119 to Korean Patent Application No. 2002-76868, filed on Dec. 5, 2002, in the Korean Intellectual Property Office, the contents of which are incorporated herein by reference in its entirety.
1. Field of the Invention
The present invention relates to a microorganism for producing riboflavin and a method for producing riboflavin using the same. More particularly, the present invention relates to a mutant of Bacillus subtilis with enhanced riboflavin productivity, when compared to the parent strain, and a method for producing riboflavin using the same.
2. Description of the Related Art
Riboflavin, also known as vitamin B2, is a water-soluble vitamin that is manufactured by biosynthesis of various microorganisms and plants. However, riboflavin cannot be biosynthesized in vertebrate including humans. Riboflavin is a precursor for flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), coenzymes involved in the oxidation-reduction reactions of all cellular bodies, and thus is an essential nutrient for animals including humans. Deficiency of riboflavin may result in inflammation of the mouth and the mucous membrane of the pharynx, skin inflammation and other skin injuries, conjunctivitis, amblyopia, growth inhibition, and weight loss. Therefore, riboflavin is used as a vitamin product for prevention or treatment of diseases associated with the aforementioned vitamin deficiency or as a feed additive for raising livestock. In particular, concentrated riboflavin has been used as a feed by itself. The current worldwide production of riboflavin is 3,000 tones per year, of which 75% is used for feed additives and the remainder is used for food and pharmaceuticals.
Presently, riboflavin is produced by chemical synthesis or by fermenting microorganisms. In the chemical synthesis, highly pure riboflavin is produced by a multi-step process using a precursor such as D-ribose. However, due to a high cost of the starting material, the production cost is also high for chemical synthesis. Therefore, fermentation process of riboflavin by microorganisms was developed. Microorganisms for the fermentation process may be any riboflavin-producing microorganisms that exist in nature or riboflavin-overproducing microorganisms that are transformed by a genetic engineering, chemical, or physical process. These microorganisms are cultured under an appropriate condition to produce riboflavin. The produced riboflavin is recovered from the culture.
Microorganisms widely known for riboflavin production are Saccharomyces sp. and Candida sp. belonging in the yeast group, Clostridium sp., Bacillus sp., and Corynebacterium sp. belonging in the bacteria group, and Eremothecium sp. and Ashbya sp. belonging in the fungi group.
U.S. Pat. No. 5,231,007 discloses a method for producing riboflavin using Candida famata yeast. It was reported that genetically engineered Bacillus subtilis and Corynebacterium ammoniagenes which overexpress the genes of the enzymes involved in riboflavin biosynthesis produced riboflavin of 4.5 g/l and 17.4 g/l, respectively [Perkins et al., J. Ind. Microbiol. Biotechnol., 22:8–18, 1999]. European Patent No. EP 0 821 063 discloses a method for producing riboflavin using a recombinant Bacillus subtilis. U.S. Pat. No. 5,837,528 discloses a recombinant strain of Bacillus subtilis for overproducing riboflavin obtained by introducing the rib operon into the parent strain using a recombinant technology. In addition, there are Eremothecium ashbyii and Ashbya gossypii ascomycete fungi which were reported by Windholz et al. [The Merck Index, Merck & Co., p. 1183, 1983] as microorganisms for riboflavin production. In particular, it was reported that culture of mutants of these ascomycete fungi in nutrient media containing molasses or vegetable oil as a main carbon source resulted in 15 g of riboflavin per 1 liter of a fermentation solution [Bigelis, Biotechnology, vol. 7b, p. 243, 1989]. Production of riboflavin using Ashbya gossypii is also disclosed in WO95/26406.
However, development of microorganisms with enhanced riboflavin productivity for mass-production of riboflavin is still in need.
Meanwhile, in the fermentation method using microorganism, a strain of microorganism with osmopressure resistance is being developed to obtain culture products in high concentration and and high yield. The strain of microorganism with osmopressure resistance is capable of producing culture products in high yield because its growth and metabolism are not inhibited by increase in the external osomopressure caused by excess carbon sources and accumulation of culture products. On the basis of the foregoing, the present inventors developed a strain of microorganism that produce 5′-inosinic acid in high concentration and in high yield by developing a strain of microorganism resistant to proline analogue to enhance its ability of proline biosynthesis and thereby reinforced its osmopressure resistance characteristic of the microorganism, which is disclosed in KR Patnet No. 330712.